Role of angiotensin II and angiotensin-(1–7) in diabetes-induced oxidative DNA damage in the corpus cavernosum  Narayana Kilarkaje, Ph.D., Mariam H.M. Yousif, Ph.D., Ahmed Z. El-Hashim, Ph.D., Batoul Makki, M.Sc., Saghir Akhtar, Ph.D., Ibrahim F. Benter, Ph.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 226-233 (July 2013) DOI: 10.1016/j.fertnstert.2013.02.046 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of diabetes mellitus (DM) and treatment of losartan (Los), captopril (Cp), and angiotensin-(1–7) (Ang) on histopathological changes in the corpus cavernosum (CC). The CC in the nondiabetic control (C) group showed normal structure with blue-green collagen fibers and reddish smooth muscle cells and caverns in between. Treatment of control groups with Cp, Los, or Ang did not significantly alter the normal histology of the nondiabetic control CC, although staining color was different due to variations in quantity of muscle and connective tissue fibers. The CC from the DM group shows marked degenerative changes in the smooth muscle cells. Diabetic rats treated with either Los (DM + Los) or Cp (DM + Cp) show reduced degenerative changes compared with that in the DM group and were comparable to their respective time-matched controls. Treatment of DM rats with Ang (DM + Ang) also resulted in a reduction in smooth muscle cell degeneration with only minimal degenerative changes noted compared with the DM group and was comparable to the Ang group. Masson’s trichrome; scale bar = 40 μm. Fertility and Sterility 2013 100, 226-233DOI: (10.1016/j.fertnstert.2013.02.046) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of diabetes mellitus (DM) and treatment of losartan (Los), captopril (Cp), and Ang-(1–7) (Ang) on oxidative stress assayed by estimating the total oxidant status (TOS) and total antioxidant status (TAS) levels in the corpus cavernosum (CC). Diabetes mellitus had a significant increase in TOS (A, C, and E) and a decrease in TAS (B, D, and F) compared with the nondiabetic controls (C). Treatment of diabetic rats with Los (DM + Los) significantly inhibited the DM-induced increase in TOS (A) and a decrease in TAS (B), whereas treatment with Cp (DM + Cp) significantly increased the TAS levels (D). Treatment of diabetic rats with Ang significantly inhibited the DM-induced decrease in TOS (F) and increase in TAS (E) compared with the DM group. Data are mean ± SEM (n = 6). *P<.05, **P<.01, and ***P<.001 versus C group. #P<.05, ##P<.01, ###P<.001- DM versus DM + drug-treated groups. Fertility and Sterility 2013 100, 226-233DOI: (10.1016/j.fertnstert.2013.02.046) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of diabetes mellitus (DM) and treatment of losartan (Los), captopril (Cp), and Ang-(1–7) (Ang) on oxidative DNA damage quantified by 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) immunohistochemistry (A) and ELISA (B, C, and D). The corpus cavernosum (CC) of the control group (C) shows very low intensity for 8-oxo-dG, localized mainly to cytoplasm (A), and low levels detected by ELISA (B, C, and D). Treatment of control group rats with Los, Cp, or Ang did not significantly alter 8-oxo-dG levels compared with vehicle control. The DM group showed significantly higher levels of the oxidized base, observed in both the cytoplasm and nuclei (A) and as measured by ELISA (B, C, and D), than the control group. Treatment of DM with either Cp (DM + Cp) or Los (DM + Los) resulted in a significant reduction in 8-oxo-dG and was limited mainly to the cytoplasm (A) and as measured by ELISA (B and C). Treatment of DM with Ang (DM + Ang) also resulted in a significant reduction in the levels of 8-oxo-dG (A and D). Data are mean ± SEM (n = 6). ***P<.05 versus C group. ###P<.001- DM versus DM + drug-treated groups. Counterstained with hematoxylin; scale bar = 40 μm. Fertility and Sterility 2013 100, 226-233DOI: (10.1016/j.fertnstert.2013.02.046) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 A schematic diagram summarizing the results on the effects of angiotensin (Ang) II and Ang-(1–7) on diabetes mellitus (DM)-induced oxidative stress in the corpus cavernosum (CC). The Ang II levels may be increased in the CC in rats with DM. This resulted in the induction of oxidative stress by increasing reactive oxygen species/reactive nitrogen species generation and decreasing antioxidant levels leading to histologic changes in smooth muscle (SM) and damage to the cytoplasmic and nuclear DNA in the CC. Treatment with Ang-(1–7) results in inhibition of the DM-induced oxidative stress and histopathological changes. TAS = total antioxidant status; TOS = total oxidant status. Fertility and Sterility 2013 100, 226-233DOI: (10.1016/j.fertnstert.2013.02.046) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions